EMBO/SourceData huggingface dataset:
Figure 1d. Real-time reverse transcription quantitative PCR (RT-qPCR) showing Fbxw7, Pten and Trp53 expression in mouse uteri at 4 weeks and 8 weeks postnatal age (NB Trp53R172H uteri were not analysed at the 4 week timepoint). Box extends from 25th to 75th percentiles, with line at median, whiskers indicate minimum and maximum values, and dots individual values (n=5 samples per group with exception of Ptendel and Ptendel, Fbxw7mut genotypes at 8 weeks which contain n=7 samples each). Statistical comparison between WT controls and other genotypes was performed by unadjusted, unpaired Mann Whitney test; * indicates P<0.05, ** indicates P<0.01e. Western blot of mouse uteri at 8 weeks postnatal age (NB Trp53R172H uteri were not analysed by Western blotting). Values indicate densitometric units (mean ± SD) normalised to control group of WT samples in case of Fbxw7 protein and both WT and Fbxw7mut samples in case of Pten and p53 proteins. Image is representative of duplicate technical replicates. Statistical comparison between controls and other groups was performed by unadjusted, unpaired Mann Whitney test; and are shown next to densitometry results where statistically significant.f. Immunohistochemical (IHC) analysis of mouse uteri at 8 weeks postnatal age. Images are representative of minimum of 3 biological replicates for each genotype and protein. Scale bar indicates 100μm.
Figure 2a. Kaplan-Meier curves showing survival of genetically engineered mouse models (GEMM) of indicated genotypes and group sizes. Statistical comparison between groups was performed by unadjusted log-rank test.c. Low and high power images of GEMM uteri at 8 weeks age and at study endpoint after H&E staining or IHC for cytokeratin 8 (CK8) (epithelia). Scale bars in low and high magnification panels indicate 1000μm and 100μm respectively. Images are representative of a minimum of 5 mice per genotype per timepoint, with exception of wild-type and Fbxw7mut mice at 8 weeks age where 3 mice were analysed.e. H&E-stained sections of high grade external sarcomas in Trp53del, Trp53del; Fbxw7mut, Trp53mut and Trp53mut; Fbxw7mut mice. Images are representative of minimum of 3 mice examined for each genotype. Scale bar indicates 100μm.
Figure 3a. (Left panel) Schematic illustrating gene expression profiling of mouse uterine samples of indicated genotypes and group sizes at 8 weeks age, with groups compared by enrichment analysis. (Right panel) Dot plot indicating enrichment of MSigDB C6 oncogenic gene sets in Ptendel; Fbxw7mut uteri vs Ptendel uteri. Arrows indicate enrichment of LEF1 signatures.
Figure 4b. Dot plot showing enrichment analysis of 1,064 putative FBXW7 substrates against GO biological processes.
Figure 5a. HEK293T cells were transfected with constructs encoding FLAG-V5-tagged FBXW7α and Myc-tagged LEF1. FLAG-V5-tagged FBXW7 α was immunoprecipitated (IP) from cell extracts with anti-FLAG antibody before immunoblotting for tagged proteins as indicated (upper panels). Lower panels show inputs. b. HEK293T cells were transfected with constructs encoding FLAG-V5-tagged FBXW7α and Myc-tagged TCF7L2. IP was performed as in (a) before immunoblotting for tagged proteins as indicated. Data information: All images shown are representative of a minimum of two independent experimentsg. HEK293T cells were transfected with Myc-tagged FBXW7α and either FLAG-V5-tagged full length LEF1 (FL) or truncated LEF1 in which the N terminal domain required for β-catenin binding has been deleted. Immunoblotting for indicated proteins was performed following IP by anti-FLAG antibody. Data information: All images shown are representative of a minimum of two independent experiments
Figure 6a. Dot plot showing log2-transformed expression of FBXW7, LEF1 and other wnt pathway genes in endometrial epithelial cell subsets defined by single cell RNAseq (scRNAseq)b. Representative LEF1 immunohistochemistry in GEMM uteri from mice of indicated genotypes at 8 weeks age. For each of the four columns (i.e genotypes), upper and lower images are taken from littermate females housed in the same cage. Images are representative of 3-4 mice as shown in (c). Scale bar indicates 100μm.c. Percentage of mice with epithelial LEF1 expression in endometria at 8 weeks age according to genotype. P value was derived from binomial logistic regression with epithelial LEF1 expression as the dependent variable.d. Immunohistochemistry for LEF1 and β-catenin from endometrial carcinomas in Ptendel ± Fbxw7mut mice. High power images show representative staining in LEF1 negative and positive glands. Scale bar bar indicates 1000μm.
Figure 1C, Quantification of telomeric allelic imbalances (TAI) in soft tissue and bone sarcoma.
Figure 5E-G, Ex vivo treatment of HRDhigh (NMFH-1, OH931, PM197, USZ-21_MFS2 and USZ-21_UPS1) sarcoma models compared with HRDlow (USZ-20_REA1, USZ-21_LG1, USZ-22_EMC2, USZ-20_SFT1 and USZ-21_CIC1) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin, doxorubicin and trabectedin.M-O, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin. Note that no sensitivity to olaparib in monotherapy was observed at 3 daysQ-S, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin.
Figure 6A, Immunofluorescence showing nuclear expression of the DNA damage marker γH2A.X (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination in HRDhigh and HRDlow sarcoma cell models as well as ovarian carcinoma UWB1.289 cells. B, Immunofluorescence showing RAD51 nuclear foci (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination only in the HRDlow sarcoma cell model (USZ-21_LG1). C, D, Quantification of γH2A.X (C) or RAD51 (D) nuclear intensity per cell compared to untreated control cells. E,E, Immunohistochemistry (IHC) in human tissue sections showing RAD51 (brown) expression in HRDlow but not HRDhigh sarcoma patients. Small-nucleated lymphocytes and/or proliferating cells appeared RAD51-positive in HRDhigh models.F, Immunofluorescence in human tissue sections showing RAD51 (magenta) nuclear foci in an HRDlow but not an HRDhigh sarcoma patient.H, Magnetic resonance imaging (MRI) of LMS patient since primary diagnosis. Open arrowheads point at metastatic lesions.J, RAD51 IHC in the metastatic patient's tissue. Compare RAD51 nuclear expression in normal tissue (arrowhead points at gastric gland) but not in tumorous gastric tissue.
Figure 1Analysis of ER architecture and dynamics as a function of RNF26 in U2OS cells ectopically expressing mCherry-KDEL and transfected with either control siRNA (siC) or siRNAs targeting RNF26 (siRNF26-1 or siRNF26-2). B. Time-lapse imaging. Top panels: representative confocal fluorescence images of Cherry-KDEL (white) at the start of time-lapse t=0 (top). Middle panels: time color coded overlays of 60x1s frames, each assigned a different color LUT (white - same ER location across all frames; color - ER movement between frames). Bottom panels: overlays of twice eroded ER sheet masks (magenta) with mCherry-KDEL signal (green). Masks were generated using ImageJ software. Zoom-ins show select PN and PP regions. Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively.I. CLIMP63 abundance as a function of RNF26. Top: representative immunoblot of U2OS cells transfected with the indicated siRNAs stained against endogenous CLIMP63 and Vinculin (loading control). Bottom: quantification of CLIMP63 abundance, normalized to Vinculin and expressed relative to control (siC=1.0), n=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Significance was assessed using Student's t-test with error bars reflecting +/- SD)J, K. Effect of RNF26 depletion on basal ER stress using IRE1-mediated XBP1 splicing as a readout. J. Relative abundance of RNF26 transcript normalized to GAPDH; n=3 independent experiments. All transcripts were detected by qPCR. Plots report values relative to control (siC=1.0). K. Quantification of IRE1-dependent XBP1 splicing. XBP-s and XBP1 transcripts isolated from U2OS cells transfected with the indicated siRNAs were detected by qPCR, normalized to GAPDH, and expressed as ratio, n=3 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Significance was assessed using Student's t-test J and K with error bars reflecting +/- SD) p<0.05; *** p<0.001, ns: not significant.
Figure 2C, D. Proximity biotinylation of endogenous vimentin by 2HA-TurboID-RNF26, catalytically inactive point mutant (I382R), or RING domain truncation mutant (∆RING) in HeLa cells following 30 min incubation with biotin prior to lysis. C. Representative immunoblots of neutravidin precipitates and lysate inputs stained against HA and vimentin. D. Quantifications of vimentin biotinylation by 2HA-TurboID-RNF26 and mutants normalized to auto biotinylated HA-tagged RNF26 species and expressed relative to RNF26 WT, n=6 independent experiments performed in U2OS and HeLa cells. Data information: WCL: whole cell lysate. Graphs in D report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.E. In vitro co-precipitation of purified recombinant Rhodamine-labeled RING-I382R (304-433) with purified recombinant His-tagged vimentin on Ni-NTA beads. Rhodamine in-gel fluorescent scan and immunoblot against vimentin are shown, representative of three independent experiments.G. Proximity-based biotinylation of endogenous vimentin by 2HA-TurboID-I382R, -∆RING, and -I382R-∆C lacking amino acids 423-433. Immunoblots of neutravidin precipitates and lysate inputs against HA and vimentin are shown and are representative of three independent experiments. Data information: WCL: whole cell lysate.H. In vitro peptide binding assay. Rhodamine-linked C-terminal peptide of RNF26 (Rho-RGILQTLNVYL) or free Rhodamine were incubated at 10nM with increasing concentrations of His-vimentin and binding was measured using fluorescence polarization; n=3 independent experiments. Data information H report mean (red line) and SEM (error bars) of sample values (open circles). Significance was assessed using Student's t-test; * p<0.05; ** p<0.01; *** p<0.001.
Figure 3A-E. Analysis of RNF26 sequence determinants with respect to the intracellular distribution of RNF26 and its colocalization with vimentin filaments. A. Representative confocal fluorescence images of U2OS cells ectopically expressing RFP-RNF26 (green) wild-type (WT) or mutants (I382R, ∆RING, ∆C), fixed and immunostained against endogenous vimentin (magenta) and VAP-A (magenta). Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions. B. Colocalization (Manders') analysis of RFP-RNF26 and mutants overlap with vimentin. Plot reports n WT=38, n I382R=31, n ΔRING=34, n ΔC=22 cells analysed from three independent experiments. C. Colocalization (Manders') analysis of RFP-RNF26 and mutants with VAP-A. Plot reports n WT=29, n I382R=21, n ΔRING=17, n ΔC=16 cells analysed from three independent experiments. D. Perinuclearity of RFP-RNF26 as a function of its RING domain determinants. Plot reports n WT=72, n I382R=48, n ΔRING=37, n ΔC=28 cells analysed from three independent experiments. E. Intracellular distribution of vimentin as a function of RNF26 (mutant) overexpression. Plot reports n WT59, n I382R=58, n ΔRING=36, n ΔC=32 technical replicates from three independent experiments. Values capped at 10. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Statistical analyses were performed using Students' T-test (B, D error bars indicate mean -/+ SD) or Mann-Whitney U test (C, E error bars indicate median and 95% confidence interval); *** p<0.001, ns: not significant.F, G. Analysis of vimentin cytoskeleton distribution as a function of RNF26. F. Representative confocal fluorescence images of control cells, cells silenced for RNF26 (si#1), or RNF26 KO cells. Zoom-ins highlight PN regions. G. Perinuclearity of vimentin in the presence or absence of RNF26. Plot reports n control=98, n siRNF26=132, n RNF26 KO=41 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Statistical analyses were performed using Students' T-test Mann-Whitney U test error bars indicate median and 95% confidence interval); *** p<0.001, ns: not significant.H. Vimentin abundance as a function of RNF26. Top: representative immunoblot of U2OS cells transfected with siRNF26#1 stained against endogenous vimentin and Vinculin (loading control). Bottom: quantification of vimentin abundance, normalized to Vinculin and expressed relative to control (siC=1.0), n=4 independent experiments. Statistical analyses were performed using Students' T-test error bars indicate mean -/+ SD) ; *** p<0.001, ns: not significant.
Figure 5Effect of vimentin ablation on ER organization and dynamics. Analysis of parental versus vimentin KO#1 U2OS cells ectopically expressing mCherry-KDEL. D. Time-lapse imaging. Left panels: representative confocal fluorescence images of Cherry-KDEL (white) at the start of time-lapse t=0 (top). Middle panels: time color coded (TCC) overlays of 60x1s frames. Right panels: overlays of twice eroded ER sheet masks (magenta) with mCherry-KDEL signal (green). Masks were generated using ImageJ software. Zoom-ins show select PN and PP regions. Scale bar = 10µm.
Figure 6D, Consequences of RNF26, UBE2J1, and vimentin loss on ERQC formation. D. Representative confocal images of fixed U2OS cells transfected or genetically modified as indicated, treated in the absence (-) or presence (+) of tunicamycin (5ug/mL, O/N), fixed and immunostained against endogenous calnexin (white).F, G. Consequences of RNF26, UBE2J1, and vimentin loss on induction of PERK-mediated UPR signaling by tunicamycin. F. U2OS cells, transfected or genetically modified as indicated, were treated in the absence (-) or presence (+) of tunicamycin (2.5µg/mL, 5hrs). Immunoblots of whole cell lysates against endogenous PERK, phospho-eIF2a, ATF4, HERP1, and Vinculin (loading control) are shown, representative of three independent experiments. G. CHOP transcripts isolated from U2OS cells transfected or genetically modified as indicated and treated in the absence (-) or presence (+) of tunicamycin (2.5ug/mL, O/N) were detected by qPCR and normalized to GAPDH; n siC (-)=4, n siC (+)=4, n siRNF26#1 (+)=3, n siRNF26#2 (+)=4, n PARENTAL (+)=4, n Vim KO (+)=4 independent experiments. Data information: Graphs report mean or median (red line) of sample values (open circles). Statistical analyses were performed using Students' T-test G error bars indicate mean -/+ SEM) p<0.05, ** p<0.01, *** p<0.001.
Figure 7A, Analysis of localization of RNF26 sequence variants and HERP1 or HERP2 in the ER. A. Representative confocal images of U2OS cells transfected with GFP-RNF26 WT, RFP-RNF26 I382R, GFP-RNF26 ΔRING and HERP1 or GFP-RNF26 WT with HA-HERP2 that were stained for HA and endogenous VAP-A. Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.C. Analysis of determinants for RNF26 complex formation with ERQC proteins HERP1 and HRD1. U2OS cells were transfected with RFP-RNF26 variants (WT, I382R, ΔRING) or EV with either HA-HERP1 or HA-HRD1 before lysis in 1% DMNG detergent, pulldown with RFP-TRAP beads and analysis by SDS-PAGE and WB. Representative images of three independent replicates.D, E. Analysis of localization of ERQC domains with vimentin. D. Representative confocal images of U2OS cells transfected with HA-HERP1 that were stained for endogenous vimentin. Indicated single channels and their corresponding color overlays are shown. Zoom-ins highlight PN regions where vimentin surrounds HERP1-labeled ERQC domains. E. Line graph analysis of HA-HERP1 (magenta) and vimentin (green) signals from E (bottom zoom image). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.F, G. Analysis of HERP1-induced ERQC domain localization as a function of vimentin and RNF26. F. Representative confocal fluorescence images of control cells, RNF26 KO cells, or vimentin KO#2 cells, transfected with HA-HERP1 and immunostained for HA. Zoom-ins highlight PN regions. G. Perinuclearity analysis of HA-HERP1 in the presence or absence of RNF26 or vimentin. Plot reports median (red line) and 95% confidence interval (error bar) of sample values (open circle), n Parental=79, n R26 KO=51, n Vim KO=45 technical replicates from three independent experiments (Mann-Whitney U test; ** p<0.01, *** p<0.001). Graphs report median (red line) and 95% confidence interval (error bars) of sample values (open circles). Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm.
Figure 8C, D. Lysosomal processing of Sec62-HALO-GFP over time in the presence or absence of RNF26 or vimentin. C. Parental, Vim KO#2 or RNF26 KO U2OS cells were transfected with Sec62-HALO-GFP overnight, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with TAMRA HALOtag ligand (200nM) for 1hr, and incubated with 6-chlorohexanol with or without Bafilomycin A1 (100nM) for the indicated times before cell lysis and analysis by SDS-PAGE. Shown is a representative in-gel fluorescence scan with indication of full-length Sec62, protease-resistant HALO fragment and molecular weight markers (MW), as well as a Vinculin blot (loading control) D. Quantification of Sec62 accumulation in LEs from (B). Unprocessed and protease-resistant (lysosomal) band intensities in (B) were calculated with ImageJ and ratios of lysosomal Sec62 versus unprocessed Sec62 were normalized to transfer in parental cells after 10 hours. Error bars show mean +/- SD of n Parental=6, n R26 KO=4, n Vim KO =6 independent experiments. Scale bar = 10µm. Graphs report mean (in D of sample values (open circles). Statistical analyses were performed using Students' T-test (D (paired) ; * p<0.05, ** p<0.01, *** p<0.001).E, F. Sec62-mediated transfer of ER to lysosomes as a function of RNF26 or vimentin. E. Representative Airyscan images of Parental, RNF26 KO or Vim KO#2 U2OS cells that were transfected with Sec62-HALO-GFP, incubated with 6-chlorohexanol for 15 minutes, washed 3 times with PBS, pulsed with JF646 HALOtag ligand for 1hr, and incubated with 6-chlorohexanol and Bafilomycin A1 for 8hrs before fixation and immunostaining for CD63 to visualize LEs. F. Colocalization (Manders') analysis of CD63 over HALO signals from (D). Plot reports n Parental=106, n R26 KO=69, n Vim KO =68 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, respectively. Scale bar = 10µm. Graphs report mean (in F) of sample values (open circles). Statistical analyses were performed using Students' T-test F (unpaired) ; * p<0.05, ** p<0.01, *** p<0.001).G, H. Sec62-mediated transfer of ER to lysosomes as a function of RNF26 overexpression and activity. G. Representative spinning disc live cell images of RNF26 WT and I382R inactive mutant with Sec62. U2OS cells were transfected overnight with Sec62-HALO-GFP and RFP-RNF26 WT or I382R, incubated for 5hrs with JF646 and imaged by spinning disc microscopy. Images show overlay of GFP (green), JF646 (magenta) and RFP signals, zoom-ins of overlay and single channels that show transfer of RNF26 into LEs (WT RNF26). H. Analysis of Sec62 transfer to proteolytic compartments as a function of RNF26 WT or I382R overexpression. Number of GFP-negative/HALO-positive signals was manually counted from cells in (B) and (G). Plot reports n EV=86, n R26 WT=35, n R26 IR =40 technical replicates from three independent experiments. Data information: Cell and nuclear boundaries are demarcated using dashed and continuous lines, median (in H) of sample values (open circles). Mann-Whitney U test (H); * p<0.05, ** p<0.01, *** p<0.001).
Figure 1B, BrdU incorporation assay for H460 and A549 cells. The mean ± SD represents 6 biological replicates in one experiment. Three independent experiments were performed. **p < 0.01, ****p < 0.0001, unpaired two-tailed Student's t test.D, Immunoblot showing the expression of Cleaved-PARP, Cleaved-CASP3, CASP3 and Actin in H460 and A549 cells.E, H460 and A549 cells were cultured in 5% Matrigel for 10 days. Representative images of the 3D Matrigel culture are shown in the top panel, and immunofluorescence staining with anti-laminin5 and DAPI is shown in the bottom panel (laminin5, red; DAPI, blue). Scale bars, 200 μm. The right panel shows the percentages of colonies with a diameter ≥ 50 μm and colonies with a diameter < 50 μm. The mean ± SD represents 5 visual fields from one experiment. Three independent experiments were performed. ****p < 0.0001, unpaired two-tailed Student's t test.F, The relative cytoplasmic nucleosomes index was measured by detection with the cell death detection ELISA kit for H460 and A549 cells after 24 h under attached or suspended conditions. Immunoblot showing the expression of Cleaved-PARP, Cleaved-CASP3, CASP3 and Actin in H460 and A549 cells after 24 h under attached or suspended conditions. The mean ± SD represents 4 biological replicates in one experiment. Three independent experiments were performed. ns, nonsignificant, **p < 0.01, unpaired two-tailed Student's t test.G, The indicated cells were allowed to grow in soft agar for 2 weeks, and colonies were counted. The mean ± SD represents 3 biological replicates in one experiment. Three independent experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student's t test.J, The volcano plot maps based on RNA-seq data show changes in the gene expression profile in H460 (C/C) cells versus H460 (T/T) cells.
Figure 2A-C, H460 (T/T), H460 (C/C), A549 (T/T) and A549 (C/C) cells were subcutaneously inoculated into BALB/c nude mice (n = 5). Primary tumors were resected after 5 weeks (A). The graphs show the mean tumor volumes (B) and weight (C). Values shown are means ± SD obtained from animal experiments. *p < 0.05, **p < 0.01, two-way ANOVA (B) and unpaired two-tailed Student's t test (C).D, IHC staining for Ki67 in primary tumors from animals. The panels show representative images (left), and the graph shows the quantification of Ki67-positive cells in the tumor (right). Scale bars, 100 μm. The mean ± SD represents 5 visual fields from each tumor tissue. ***p < 0.001, ****p < 0.0001, unpaired two-tailed Student's t test.F, The scatter plot and the percentage of HLA-I-positive circulating tumor cells (CTCs) in the peripheral blood was analyzed by Flow Cytometry, and numerical statistics of the two groups were calculated (n = 5). Tumor-free mice were used as a negative control to determine the gating. *p < 0.05, **p < 0.01, unpaired two-tailed Student's t test.I, The log-rank (Mantel-Cox) test of overall survival of 239 Chinese lung cancer patients based on rs1663689 genotype.